US7118896B2 - Methods and compositions for modification of lipid biosynthesis - Google Patents

Abstract

The invention provides polypeptides having wax ester synthase and acyl-CoA:diacylglycerol acyltransferase activity. Also provided are the nucleic acids encoding such polypeptides, cells and organisms transformed therewith and methods of use thereof. The invention allows the modification of lipid profiles in host cells and organisms. Novel methods for the production of waxy esters are also provided by the invention.

Description

This application claims priority to U.S. Provisional Patent Application Ser. No. 60/360,774, filed on Mar. 1, 2002, the entire disclosure of which is specifically incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to newly identified and isolated polynucleotides, proteins encoded by the polynucleotides, methods for producing proteins, and uses for the polynucleotides and proteins. More specifically, the current invention relates to bifunctional wax ester synthase proteins, polynucleotides encoding the proteins, and methods and compositions related thereto.

2. Description of Related Art

The capability for biosynthesis of neutral lipids is widely distributed in nature and is found in animals, plants as well as microorganisms. In bacteria, the most abundant class of neutral lipids are polyhydroxyalkanoic acids serving as intracellular carbon and energy storage (Steinbüchel, 1991), but also few examples of substantial triacylglycerol (TAG) accumulation have been reported for species mainly belonging to the actinomycetes genera Mycobacterium (Barksdale and Kim, 1977), Nocardia and Rhodococcus (Alvarez et al., 1997) and Streptomyces (Olukoshi and Packter, 1994). Furthermore, biosynthesis of wax esters (oxoesters of long-chain primary fatty alcohols and long-chain fatty acids) has been frequently reported for members of the genus Acinetobacter (Fixter et al., 1986).

TAGs are the dominating storage lipid in animals, plants and eukaryotic microorganisms. TAG biosynthesis is involved in animals in numerous processes such as regulation of plasma TAG concentration, fat storage in adipocytes and milk production (Bell and Coleman, 1980). In plants, TAG synthesis is mainly important for the generation of seed oils (Lassner, 1997) Using diacylglycerol (DAG) as a substrate, three different classes of enzymes are known mediating TAG formation (Lehner and Kuksis, 1996). Acyl-CoA:DAG acyltransferase (DGAT) catalyzes the acylation of DAG using acyl-CoA as a substrate. Two DGAT families designated as DGAT1 and DGAT2 are known, which exhibit no sequence homologies to each other. Members of the DGAT1 gene family occur in animals and plants (Cases et al., 1998; Hobbs et al., 1999; Routaboul et al., 1999; Zou et al., 1999), whereas members of the DGAT2 gene family were found in animals (Cases et al., 2001), plants (Bouvier-Navée et al., 2000) and yeasts (Oelkers et al., 2002). In human, one DGAT1 related gene and five DGAT2 related genes were identified (Cases et al., 2001).

Recently, DGAT has attracted great interest since it is a potential therapeutical target for obesity treatment (Chen and Farese Jr., 2000). Acyl-CoA-independent TAG synthesis is mediated by a phospholipid:DAG acyltransferase found in yeast and plants, which uses phospolipids as acyl donors for DAG esterification (Dahlqvist et al., 2000). A third alternative mechanism present in animals and plants is TAG synthesis by a DAG-DAG-transacylase which uses DAG as acyl donor and acceptor yielding TAG and monoacylglycerol (Lehner and Kuksis, 1993; Stobart et al., 1997), but no gene coding such a transacylase could be identified yet.

Linear wax esters are lipophilic compounds containing a long chain fatty alcohol esterified to a long chain fatty acid. These wax esters are found in a number of diverse organisms ranging from mammals to plants to bacteria. For instance, wax esters are the principal component of spermaceti oil which, until recently, was obtained from the head cavity of sperm whales. Since the world-wide ban on whale hunting, however, the only natural source of wax esters on a commercial scale has been the seeds of jojoba, a bush or shrub that is adapted to growth in hot arid habitats. In jojoba plants, waxes are stored in the seeds of the plant where they serve as a means of energy storage for developing seedlings. Wax esters have also been found in several species of bacteria such as Acinetobacter calcoaceticus, a gram negative aerobic bacteria that accumulates wax esters when grown under nitrogen limited conditions. Wax esters from these bacterial sources, however, have not been utilized on a commercial scale.

Wax esters have a multitude of important commercial applications in a variety of technical areas, including the medical, cosmetics and food industries as well as their more traditional usage as lubricants for mechanical parts and the like. The wax esters obtained from jojoba can replace sperm whale oil in most or all traditional uses. They are useful for applications in cosmetics, as a lubricant, as an additive for leather processing, as a carrier for pharmaceuticals and as a solvent. Hydrogenation of the wax to eliminate double bonds produces a hard wax which is useful for surface treatments, in textile sizing, in coating paper containers and in cosmetics (e.g., lipstick and creams). Sulphurization of the wax or other modifications make the substance useful in specialty lubricant applications, as a textile softener, as a component of printing inks, and as a component in many technical products such as corrosion inhibitors, surfactants, detergents, disinfectants, plasticizers, resins and emulsifiers. For some of these applications the fatty alcohol derived by hydrolysis of the wax ester is the most valuable ingredient derived from the wax ester.

Because the yield of the jojoba plant is extremely low, however, the oil is relatively expensive compared with edible oils from plants or technically comparable materials from petroleum and its use has been limited to cosmetic products. Thus, a need exists to develop an alternate biological source of wax esters. One possibility, in this respect, is to recombinantly engineer a microbial species for efficient production of wax esters. Toward that end, information concerning enzymes and enzymatic pathways which are involved in wax ester biosynthesis, and the nucleic acid sequences that encode these enzymes are needed.

The most detailed information concerning wax ester biosynthesis concerns wax biosynthesis in jojoba plants, where it appears that two enzymes catalyze the formation of wax esters. The first step of the pathway is catalyzed by a fatty acyl-CoA reductase which reduces very long chain fatty acyl CoA (a very long chain fatty acyl CoA generally having greater than 18 carbons), and is known to catalyze the formation of a long chain alcohol directly from this substrate via an aldehyde intermediate. The second enzyme (wax ester synthase), an acyl-CoA-fatty alcohol transferase catalyzes the formation of an ester linkage between acyl-CoA and a fatty alcohol to yield a wax ester.

The pathway of wax ester biosynthesis in A. calcoaceticus, in contrast to the jojoba plant, comprises three enzymatic steps involved in the conversion of long-chain acyl-CoA to wax esters. In the first step, acyl-CoA is reduced by an NADPH-dependent acyl-CoA reductase to the corresponding fatty aldehyde. In the second step, the aldehyde is further reduced to the corresponding fatty alcohol catalyzed by the fatty aldehyde reductase. Finally, an acyl-CoA:fatty alcohol acyl transferase (wax ester synthase) condenses the fatty alcohol with acyl-CoA resulting in the formation of the wax ester.

Wax ester synthesis and WS activity have been reported for M. tuberculosis (Wang et al., 1972), and TAG accumulation and DGAT activity have been shown for M. smegmatis (Nakagawa et al., 1976; Wun et al., 1977). However, no proteins or genes have been reported to which these activities could be attributed.

Irrespective of the species involved, therefore, a key enzymatic step involved in wax ester biosynthesis is the transfer of an acyl chain from fatty acyl-CoA to a fatty alcohol, and this reaction is catalyzed by wax ester synthase. While several wax ester synthases have been described in terms of their substrate specificities and intracellular locations, very little is known about the proteins associated with this activity and the genes encoding this enzyme. In fact, the only gene encoding a wax ester synthase that has been identified is from jojoba. Thus, a need exists to identify genes encoding wax ester synthases from other species. In particular, a need exists to identify genes encoding wax ester synthases from a species that could be engineered to produce wax esters in large quantities and at a relatively affordable cost. The present invention addresses this need by providing polynucleotide sequences encoding bacterial wax ester synthases.

SUMMARY OF THE INVENTION

In one aspect, the invention provides an isolated polynucleotide encoding a polypeptide having wax ester synthase/DGAT activity and having a nucleotide sequence at least about 60% homologous to SEQ ID NO:1. In certain embodiments of the invention, an isolated polynucleotide in accordance with the invention comprises a polynucleotide that hybridizes to SEQ ID NO:1 under conditions of 5×SSC, 50% formamide and 42° C., and which encodes a protein having the same biological function. Such polynucleotides may encode the same polypeptide as SEQ ID NO:1.

In another aspect of the invention provides a recombinant vector comprising a polynucleotide of the invention. In certain embodiments, the recombinant vector may comprise the polynucleotide linked to one or more regulatory regions and/or any other desired elements.

In yet another aspect, the invention provides a protein or polypeptide fragment encoded by the polynucleotide of SEQ ID NO:1 or a fragment thereof having wax ester synthase/DGAT activity. In certain embodiments of the invention, a protein comprising the amino acid sequence of SEQ ID NO:4 or a fragment of SEQ ID NO:4 is provided, wherein said fragment has wax ester synthase activity. In certain embodiments of the invention, the protein and/or fragment may have wax ester synthase and DGAT activity. In certain further embodiments of the invention, a protein or polypeptide may comprise one or more of the following amino acid sequences: HHAXVDGV (SEQ ID NO:16), NDVVLA (SEQ ID NO:17), GALRXYL (SEQ ID NO:18), PLXAMVP (SEQ ID NO:19), ISNVPGP (SEQ ID NO:20), REPLYXNGA (SEQ ID NO:21), including one, two, three, four, five or all six of these amino acid sequences.

In still yet another aspect of the invention, a recombinant construct is provided comprising a polynucleotide sequence encoding a conserved polypeptide fragment with wax ester synthase and/or DGAT activity, and further wherein the polypeptide comprises at least one of the following amino acid sequences selected from the group consisting of HHAXVDGV (SEQ ID NO:16), NDVVLA (SEQ ID NO:17), GALRXYL (SEQ ID NO:18), PLXAMVP (SEQ ID NO:19), ISNVPGP (SEQ ID NO:20), REPLYXNGA (SEQ ID NO:21), including one, two, three, four, five or all six of these amino acid sequences. In one embodiment of the invention, the polynucleotide sequence is a bacterial sequence. In still yet another embodiment is provided a host cell containing this recombinant construct. In one embodiment of the invention, the host cell may be any type of cell.

In still yet another aspect, a method is provided a method for producing a wax ester comprising culturing a host cell described above under conditions permitting expression of the polypeptide having wax ester synthase activity. In certain embodiments of the invention, the polypeptide may have DGAT activity. The method may comprise culturing the cell in one or more substrates for the waxy ester synthase/DGAT. The wax ester may or may not be isolated from the host cell or any media in which the host cell is cultured.

In still yet another aspect, the invention provides a method of modifying accumulation of wax esters in a host cell that comprises transforming a host cell with a recombinant construct of the invention. In certain embodiments, the recombinant construct may comprise a regulatory sequence operably linked to a nucleic acid sequence, said nucleic acid sequence encoding a polynucleotide encoding a polypeptide having wax ester synthase and/or DGAT activity or a fragment thereof, and culturing said host cell under conditions wherein said host cell expresses a polypeptide having wax ester synthase and/or DGAT activity such that said host cell has a modified wax ester composition compared to host cells without the recombinant construct. In certain embodiments of the invention, the method comprises increasing the wax ester content of a host cell.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, appended claims, and accompanying figures, where:

FIG. 1 shows the results of a thin layer chromatography (“TLC”) analysis of miniTn10Km-induced mutants from A. calcoaceticus BD413 (Ndx^r) after cultivation under storage conditions. TAG, triolein standard; WAX, cetylpalmitate standard.

FIG. 2 shows the restriction pattern of the miniTn10Km-harboring 8.4-kbp EcoRI fragment isolated from wax⁻mutants of A. calcoaceticus BD413 (Ndx^r).

FIG. 3 shows the molecular organization of the 6.9-kbp EcoRI-fragment harboring the wax open reading frame from A. calcoaceticus BD413. The insertion locus of miniTn10Km is indicated by the triangle.

FIG. 4 shows the influence of the wax gene on storage lipid accumulation in A. calcoaceticus BD413. Cells were cultivated under storage conditions for 24 h. Lane A, TAG; Lane B, hexadecanol; Lane C, hexadecanal, Lane D, cetylpalmitate; Lane 1, A. calcoaceticus BD413; Lane 2, A. calcoaceticus BD413waxΩKm; Lane 3, A. calcoaceticus BD413 (pSER200-4); Lane 4, A. calcoaceticus BD413 (pSER200-4:wax).

FIG. 5 is the nucleotide sequence of the PCR™-amplified 1.9-kbp fragment from A. calcoaceticus BD413 comprising the wax/dgat gene (SEQ ID NO:3). The wax gene is shown in bold face. A putative ribosome binding site is double-underlined. The EcoRI and BamHI restriction sites used for cloning are underlined.

FIG. 6 is the nucleotide sequence of the PCR™-amplified 1.47-kbp wax/dgat gene from A. calcoaceticus BD413 (SEQ ID NO:2). The wax gene is shown in bold face. The linked ribosome binding site (Shine/Dalgarno sequence) for E. coli is double-underlined. The EcoRI and BamHI restriction sites used for cloning are underlined.

FIG. 7 is the recombinant wax ester biosynthesis in P. citronellolis. Cells were cultivated and analyzed by TLC as described in Materials and methods. Wax, cetylpalmitate standard; Lane 1: P. citronellolis (pBBRIMCS-2) cultivated with 0.3% (w/v) hexadecanol; Lane 2: P. citronellolis (pBBRIMCS-2) cultivated with 0.3% (w/v) hexadecanol plus 0.5% (w/v) gluconate; Lane 3: P. citronellolis (pBBRIMCS-2:wax) cultivated with 0.3% (w/v) hexadecanol; Lane 4: P. citronellolis (pBBRIMCS-2:wax) cultivated with 0.3% (w/v) hexadecanol plus 0.5% (w/v) gluconate.

FIG. 8 depicts the polynucleotide sequence of SEQ ID NO:1.

FIG. 9 depicts the deduced amino acid sequence of SEQ ID NO:4.

FIG. 10 shows an alignment of the polypeptide sequence encoded by SEQ ID NO:1 versus deduced bacterial sequences (SEQ ID NOs:24–31). Conserved regions are shown by shading.

FIG. 11 shows an alignment of the polypeptide sequence encoded by SEQ ID NO:1 versus deduced Arabidopsis thaliana and bacterial sequences (SEQ ID NOs:22–31). Conserved regions are shown by shading.

FIGS. 12A, B shows properties of the bifunctional WS/DGAT. (FIG. 12A) Reactions catalyzed by the bifunctional enzyme. (FIG. 12B) Hydrophobicity plot (Kyte and Doolittle, 1982) of WS/DGAT (window size 9). A putative transmembrane domain predicted by the TMAP program is indicated by the grey bar. The black bar region exhibits some homology to a conserved condensing domain containing a putative active site.

FIGS. 13A, B, C Substrate specificities of the bifunctional WS/DGAT. Measurements were done using insoluble fraction of crude extract of E. coli XL1-Blue (pKS::wax/dgat) obtained after 30 min centrifugation at 35,000×g. Values are averages of two independent studies. (FIG. 13A) Fatty alcohol specificity of the WS reaction. (FIG. 13B) Acyl-CoA specificity of the WS reaction. (FIG. 13C) Acyl-CoA specificity of the DGAT reaction.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the initial discovery and isolation of the polynucleotide encoding a wax ester synthase (WS) protein from A. calcoaceticus, which protein also has acyl-CoA:diacylglycerol acyltransferase (DGAT) activity (e.g., WS/DGAT). Therefore, when wax ester synthase polypepetides and nucleic acids are referred to herein this specifically includes the inherent bifunctional DGAT activity. The sequence from A. calcoaceticus has been determined and is given in SEQ ID NO:1 and is also set forth in FIG. 8. Moreover, the amino acid sequence of the wax ester synthase/DGAT protein produced by the isolated nucleic acid sequence has been deduced and is given in FIG. 9 and corresponds to SEQ ID NO:4. Although a particular embodiment of the nucleotide sequence disclosed herein is given in SEQ ID NO:1, it should be understood that other biologically functional equivalent forms of the nucleic acid sequence of the present invention can be readily isolated using conventional DNA-DNA and DNA-RNA hybridization techniques. Thus the present invention also includes nucleotide sequences that hybridize to SEQ ID NO:1 or its complement under moderate to high stringency conditions and encode proteins exhibiting the same or similar biological activity as that of protein of SEQ ID NO:4 disclosed herein. Also included in the invention are polynucleotides that exhibit 90%, preferably 92%, more preferably 95% and more 98% sequence identity with SEQ ID NO:1, its complement or SEQ ID NO:2.

One advance of the current invention relates to novel methods for the production of wax esters. A strong demand exists for large-scale production of cheap jojoba-like wax esters which have multiple commercial uses. Jojoba oil is the only alternative natural source of wax esters to sperm whale oil which is used at a commercial scale, but the high production costs restrict its use currently on cosmetical applications. The jojoba WS is not functionally expressed in microorganisms like E. coli and S. cerevisiae. In contrast, the inventors have demonstrated WS/DGAT that is active in different bacterial hosts. In P. citronellolis, the heterologous expression of WS/DGAT lead to production of wax esters if a long-chain fatty alcohol was provided as carbon source which also delivers fatty acyl-CoA during catabolism by the alkane degradation pathway. By variation of the fatty alcohol used as carbon source, one may vary the composition of the produced wax esters. The invention provides the basis, for example, for microbial biotechnological production of jojoba-like wax esters. The fast growth rates, the possibility to influence the wax composition by altering the culture conditions and the easy accessibility of bacteria to genetic and metabolic engineering will support such efforts.

In certain embodiments, the invention provides nucleic acid sequences that hybridize to SEQ ID NO:1 or its complement under moderate to high stringency conditions. As is well known in the art, stringency is related to the Tm of the hybrid forrned. The Tm (melting temperature) of a nucleic acid hybrid is the temperature at which 50% of the bases are base-paired. For example, if one of the partners in a hybrid is a short oligonucleotide of approximately 20bases, 50% of the duplexes are typically strand separated at the T_m. In this case, the T_m reflects a time-independent equilibrium that depends on the concentration of oligonucleotide. In contrast, if both strands are longer, the T_m corresponds to a situation in which the strands are held together in structure possibly containing alternating duplex and denatured regions. In this case, the T_m reflects an intramolecular equilibrium that is independent of time and polynucleotide concentration.

As is also well known in the art, T_m is dependent on the composition of the polynucleotide (e.g. length, type of duplex, base composition, and extent of precise base pairing) and the composition of the solvent (e.g. salt concentration and the presence of denaturants such formamide). An equation for the calculation of T_m can be found in Sambrook, et al. (2001), and is: T_m=81.5° C.−16.6(log₁₀[Na⁺])=0.41(% G+C)−0.63(% formamide)−600/L). Where L is the length of the hybrid in base pairs, the concentration of Na⁺ is in the range of 0.01M to 0.4M and the G+C content is in the range of 30% to 75%. Equations for hybrids involving RNA can be found in the same reference. Alternative equations can be found in Davis et al. (1994).

Methods for hybridization and washing are well known in the art and can be found in standard references in molecular biology such as those cited herein. In general, hybridizations are usually carried out in solutions of high ionic strength (6×SSC or 6×SSPE) at a temperature 20–25′ C. below the T_m. High stringency wash conditions are often determined empirically in preliminary experiments, but usually involve a combination of salt and temperature that is approximately 12–20° C. below the T_m. One example of such wash conditions is 5×SSC, 50% formamide at 42° C. An example with higher stringency conditions is 1×SSC at 60° C. Another example of high stringency wash conditions is 0.1×SSPE, 0.1% SDS at 42° C. (Meinkoth and Wahl, 1984). An example of even higher stringency wash conditions is 0.1×SSPE, 0.1% SDS at 50–65° C. In one preferred embodiment, high stringency washing is carried out under conditions of 1×SSC and 60° C.

It is well known to those of ordinary skill in the art that different compositions can result in equal stringency conditions for hybridization depending on well known factors such as the concentration of Na⁺, the % formamide, the temperature, the T_m of the hybrid to be formed, and the composition of the hybrid, e.g. DNA-DNA, DNA-RNA, or RNA-RNA. Thus the invention also encompasses nucleotide sequences that hybridize under conditions equivalent to those described above.

Also included in the invention are polynucleotides that exhibit 90%, preferably 92%, more preferably 95%, and still more preferably 98% sequence identity or homology with SEQ ID NO:1, its respective complement or SEQ ID NO:2. Such nucleotide sequences preferably hybridize to the nucleic acid of SEQ ID NO:1 or its respective complement under high stringency conditions.

“Homology,” as is well understood in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, “homology” also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as determined by the match between strings of such sequences. “Homology” can be readily calculated by known methods including, but not limited to, those described in Computational Molecular Biology (1988); Biocomputing. Informatics and Genome Projects (1993); Computer Analysis of Sequence Data, Part I (1994); Sequence Analysis in Molecular Biology (1987); Sequence Analysis Prime (1991); and Carillo and Lipman (1988). Methods to determine homology are designed to give the largest match between the sequences tested. Moreover, methods to determine homology are codified in publicly available programs. Computer programs which can be used to determine identity/homology between two sequences include, but are not limited to, GCG (Devereux et al., 1984; suite of five BLAST programs, three designed for nucleotide sequences queries (BLASTN, BLASTX, and TBLASTX) and two designed for protein sequence queries (BLASTP and TBLASTN) (Coulson, 1994; Birren, et al., 1997). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual; Altschul et al., 1990). The well known Smith Waterman algorithm can also be used to determine homology.

The present invention also involves recombinant polynucleotides comprising the isolated protein along with other sequences. Such recombinant polynucleotides are commonly used as cloning or expression vectors although other uses are possible. A recombinant polynucleotide is one in which polynucleotide sequences of different organisms have been joined together to form a single unit. A cloning vector is a self replicating DNA molecule that serves to transfer a DNA segment into a host cell. The three most common types of cloning vectors are bacterial plasmids, phages, and other viruses. An expression vector is a cloning vector designed so that a coding sequence inserted at a particular site will be transcribed and translated into a protein.

Both cloning and expression vectors contain nucleotide sequences that allow the vectors to replicate in one or more suitable host cells. In cloning vectors, this sequence is generally one that-enables the vector to replicate independently of the host cell chromosomes, and also includes either origins of replication or autonomously replicating sequences. Various bacterial and viral origins of replication are well known to those skilled in the art and include, but are not limited to the pBR322 plasmid origin, the 2 plasmid origin, and the SV40, polyoma, adenovirus, VSV and BPV viral origins.

The polynucleotide sequence of the present invention may be used to produce proteins by the use of recombinant expression vectors containing the sequence. A great variety of expression vectors can be used to express a polypeptide of the invention. Such vectors include chromosomal, episomal and virus-derived vectors e.g., vectors derived from bacterial plasmids, from bacteriophage, from yeast episomes, from yeast chromosomal elements, from viruses such as baculoviruses, papoviruses such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses; pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. All such vectors may be used for expression in accordance with this aspect of the present invention. Generally, any vector suitable to maintain, propagate or express polynucleotides to express a polypeptide in a host may be used for expression in this regard. Therefore, any other vector that is replicable and viable in the host may be used.

The appropriate DNA sequence may be inserted into the vector by any of a variety of well-known and routine techniques. In general, a DNA sequence for expression is joined to an expression vector by cleaving the DNA sequence and the expression vector with one or more restriction endonucleases and then joining the restriction fragments together using T4-:DNA ligase. Procedures for restriction and ligation that can be used to this end are well known and routine to those of skill in the art. Suitable procedures in this regard, and for constructing expression vectors using alternative techniques, which also are well known and routine to those-of skill in the art, are set forth in great detail in Sambrook et al., (2001); Ausubel et al. (1995).

In an expression vector, the sequence of interest is operably linked to a suitable regulatory sequence, expression control sequence or promoter recognized by the host cell to direct mRNA synthesis. Promoters are untranslated sequences located generally 100 to 1000 base pairs (bp) upstream from the start codon of a structural gene that regulate the transcription and translation of nucleic acid sequences under their control. Promoters are generally classified as either inducible or constitutive. Inducible promoters are promoters that initiate increased levels of transcription from DNA under their control in response to some change in the environment, e.g. the presence or absence of a nutrient or a change in temperature. Constitutive promoters, in contrast, maintain a relatively constant level of transcription. In addition, useful promoters can also confer appropriate cellular and temporal specificity. Such promoters include those that are developmentally-regulated or organelle, tissue or cell-specific.

A nucleic acid sequence is operably linked when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operatively linked to DNA for a polypeptide if it is expressed as a preprotein which participates in the secretion of the polypeptide; a promoter is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, operably linked sequences are contiguous and, in the case of a secretory leader, contiguous and in reading frame. Linking is achieved by blunt end ligation or ligation at restriction enzyme sites. If suitable restriction sites are not available, then synthetic oligonucleotide adapters or linkers can be used as is known to those skilled in the art (Sambrook et al., 2001; Ausubel et al., 1995).

Those skilled in the art will recognize that there are a number of promoters which are functional in bacterial cells, and have been described in the literature including constitutive, inducible, developmentally regulated, and environmentally regulated promoters. Of particular interest is the use of promoters (also referred to as transcriptional initiation regions) functional in bacterial host cells. Representatives of such promoters include the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters, promoters of retroviral LTRs, the CaMV 35S promoter (Assaad and Signer, 1990), coconut foliar decay virus (CFDV) DNA (U.S. Pat. No. 6,303,345), and the endogenous promoters of P. citronellolis, to name just a few of the well-known promoters. It will be understood that numerous promoters not mentioned are suitable for use in this aspect of the invention, are well known, and may be readily employed by those of skill in the manner illustrated by the discussion and the examples herein. Other promoters known to control the expression of genes in prokaryotic or eukaryotic cells can be used and are known to those skilled in the art. Expression vectors may also contain a ribosome binding site for translation initiation, and a transcription terminator. The vector may also contain sequences useful for the amplification of gene expression.

Regulatory transcript termination regions may be provided in expression constructs of this invention as well. Transcript termination regions may be provided by the DNA sequence encoding the WS/DGAT or a convenient transcription termination region derived from a different gene source, for example, the transcript termination region which is naturally associated with the transcript initiation region. The skilled artisan will recognize that any convenient transcript termination region which is capable of terminating transcription in a host cell may be employed in the constructs of the present invention.

Expression and cloning vectors can and usually do contain a structural gene or selection marker having the necessary regulatory regions for expression in a host and providing for selection of transformant cells. The gene may provide for resistance to a cytotoxic agent, e.g. antibiotic, heavy metal, toxin, etc., complementation providing prototrophy to an auxotrophic host, viral immunity or the like. Depending upon the number of different host species into which the expression construct or components thereof are introduced, one or more markers may be employed, where different conditions for selection are used for the different hosts.

Non-limiting examples of suitable selection markers include genes that confer resistance to bleomycin, gentamycin, glypho sate, hygromycin, kanamycin, methotrex ate, nalidixic acid, phleomycin, phosphinotricin, spectinomycin, streptomycin, sulfonamide, sulfonylureas, and tetracycline. Maliga et al. (1995). Examples of markers include, but are not limited to, alkaline phosphatase (AP), myc, hemagglutinin (HA), β glucuronidase (GUS), luciferase, and green fluorescent protein (GFP). In one embodiment, the vectors contain structural genes providing resistance to kanamycin and nalidixic acid.

In addition, expression vectors can also contain marker sequences operatively linked to a nucleotide sequence for a protein that encode an additional protein used as a marker. The result is a hybrid or fusion protein comprising two linked and different proteins. The marker protein can provide, for example, an immunological or enzymatic marker for the recombinant protein produced by the expression vector.

Additionally, the end of the polynucleotide can be modified by the addition of a sequence encoding an amino acid sequence useful for purification of the protein produced by affinity chromatography. Various methods have been devised for the addition of such affinity purification moieties to proteins. Representative examples can be found in U.S. Pat. Nos. 4,703,004, 4,782,137, 4,845,341, 5,935,824, and 5,594,115. Any method known in the art for the addition of nucleotide sequences encoding purification moieties can be used, for example those contained in Innis et al. (1990); Sambrook et al. (2001). More specifically, one embodiment of the present invention provides expression constructs containing the nucleotide sequence represented in SEQ ID NO:1. Such constructs are prepared as demonstrated in the Examples below.

More particularly, the present invention includes recombinant constructs comprising the isolated polynucleotide sequence of the present invention. The constructs can include a vector, such as a plasmid or viral vector, into which the sequence of the present invention has been inserted, either in the forward or reverse orientation. The recombinant construct further comprises regulatory sequences, including for example, a promoter operatively linked to the sequence. Large numbers of suitable vectors and promoters are known to those skilled in the art and are commercially available. In one preferred embodiment, the pCS2+, the pCEP4 (Invitrogen) and the pIRESneo (Clontech) vectors are used. It will be understood by those skilled in the art, however, that other plasmids or vectors may be used as long as they are replicable and viable or capable of expressing the encoded protein in the host.

The polynucleotide sequence of the present invention can also be part of an expression cassette that at a minimum comprises, operably linked in the 5′ to 3′ direction, a promoter, a polynucleotide of the present invention, and a transcriptional termination signal sequence functional in a host cell. The promoter can be of any of the types discussed herein, for example, a tissue specific promoter, a developmentally regulated promoter, an organelle specific promoter, etc. The expression cassette can further comprise an operably linked targeting sequence, transit or secretion peptide coding region capable of directing transport of the protein produced. The expression cassette can also further comprise a nucleotide sequence encoding a selectable marker and a purification moiety.

A further embodiment of the present invention relates to transformed host cells containing the constructs comprising the polynucleotide sequence of the present invention. The host cell can be a higher eukaryotic cell, such as a mammalian or a plant cell, or a lower eukaryotic cell such as an insect cell or a yeast cell, or the host can be a prokaryotic cell such, as a bacterial cell. In one embodiment, the host cell is a bacterial cell. In one embodiment, the host cell is an E. coli cell or a P. citronellolis cell. Introduction of the construct into the host cell can be accomplished by a variety of methods including calcium phosphate transfection, DEAE-dextran mediated transfection, Polybrene mediated transfection, protoplast fusion, liposome mediated transfection, direct microinjection into the nuclei, biolistic (gene gun) devices, scrape loading, and electroporation.

The present invention also relates to proteins encoded by the isolated polynucleotides. As used herein the term protein includes fragments, analogs and derivatives of the wax ester synthase-like protein. The terms “fragment,” “derivative” and “analog” as used herein mean a polypeptide that retains essentially the same biological function or activity as the wax ester synthase/DGAT encoded by the sequence of the present invention. For example, an analog includes a proprotein which can be cleaved to produce an active mature protein. The protein of the present invention can be a natural protein, a recombinant protein or a synthetic protein or a polypeptide.

Those of ordinary skill in the art are aware that modifications in the amino acid sequence of a peptide, polypeptide, or protein can result in equivalent, or possibly improved, second generation peptides, etc., that display equivalent or superior functional characteristics when compared-to the original amino acid sequence. The present invention accordingly encompasses such modified amino acid sequences. Alterations can include amino acid insertions, deletions, substitutions, truncations, fusions, shuffling of subunit sequences, and the like, provided that the peptide sequences produced by such modifications have substantially the same functional properties as the naturally occurring counterpart sequences disclosed herein. Biological activity or function can be determined by, for example, the ability of the protein to increase wax ester production in a host cell as depicted in the examples below.

One factor that can be considered in making such changes is the hydropathic index of amino acids. The importance of the hydropathic amino acid index in conferring interactive biological function on a protein has been discussed by Kyte and Doolittle (1982). It is accepted that the relative hydropathic character of amino acids contributes to the secondary structure of the resultant protein. This, in turn, affects the interaction of the protein with molecules such as enzymes, substrates, receptors, DNA, -antibodies, antigens, etc.

Based on its hydrophobicity and charge characteristics, each amino acid has been assigned a hydropathic index as follows: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate/glutamane/aspartate/asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

As is known in the art, certain amino acids in a peptide or protein can be substituted for other amino acids having a similar hydropathic index or score and produce a resultant peptide or protein having similar biological activity, i.e., which still retains biological functionality. In making such changes, it is preferable that amino acids having hydropathic indices within 2 are substituted for one another. More preferred substitutions are those wherein the amino acids have hydropathic indices within 1. Most preferred substitutions are those wherein the amino acids have hydropathic indices within 0.5.

Like amino acids can also be substituted on the basis of hydrophilicity. U.S. Pat. No. 4,554,101 discloses that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. The following hydrophilicity values have been assigned to amino acids: arginine/lysine (+3.0); aspartate/glutamate (+3.0 1); serine (+0.3); asparagine/glutamane (+0.2); glycine (0); threonine (−0.4); proline (−0.5 1); alanine/histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine/isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); and tryptophan (−3.4). Thus, one amino acid in a peptide, polypeptide, or protein can be substituted by another amino acid having a similar hydrophilicity score and still produce a resultant protein having similar biological activity, i.e., still retaining correct biological function. In making such changes, amino acids having hydropathic indices within 2 are preferably substituted for one another, those within 1 are more preferred, and those within 0.5 are most preferred.

As outlined above, amino acid substitutions in the peptides of the present invention can be based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions that take various of the foregoing characteristics into consideration in order to produce conservative amino acid changes resulting in silent changes within the present peptides, etc., can be selected from other members of the class to which the naturally occurring amino acid belongs. Amino acids can be divided into the following four groups: (1) acidic amino acids; (2) basic amino acids; (3) neutral polar amino acids; and (4) neutral non-polar amino acids. Representative amino acids within these various groups include, but are not limited to: (1) acidic (negatively charged) amino acids such as aspartic acid and glutamic acid; (2) basic (positively charged) amino acids such as arginine, histidine, and lysine; (3) neutral polar amino acids such as glycine, serine, threonine, cysteine, cystine, tyrosine, asparagine, and glutamine; and: (4) neutral non-polar amino acids such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. It should be noted that changes which are not expected to be advantageous can also be useful if these result in the production of functional sequences.

The fragment, derivative or analog of the proteins encoded by the polynucleotide sequence of the present invention may be, for example and without limitation, (i) one in which one or more amino acid residues are substituted with a conserved or non-conserved amino acid residue, and such substituted amino acid residue may or may not be one encoded by the genetic code; (ii) one in which one or more of the amino acid residues includes a substituent group; (iii) one in which the mature protein is fused to another compound such as a compound to increase the half-life of the protein; (iv) one in which additional amino acids are fused to the protein to aid in purification or in detection and identification; or (v) one in which additional amino acid residues are fused to the protein to aid in modifying tissue distribution or localization of the protein to certain locations such as the cell membrane or extracellular compartments.

The term protein also includes forms of the protein to which one or more substituent groups have been added. A substituent is an atom or group of atoms that is introduced into a molecule by replacement of another atom or group of atoms. Such groups include, but are not limited to, lipids, phosphate groups, sugars and carbohydrates. Thus, the term protein includes, for example, lipoproteins, glycoproteins, phosphoproteins and phospholipoproteins.

The present invention also includes methods for the production of the protein of interest from cells transformed with a polynucleotide sequence of the present invention. Proteins can be expressed in mammalian cells, plant cells, insect cells, yeast, bacteria, bacteriophage, or other appropriate host cells. Host cells are genetically transformed to produce the protein of interest by introduction of an expression vector containing the nucleic acid sequence of interest. The characteristics of suitable cloning vectors and the methods for their introduction into host cells have been previously discussed. Alternatively, cell-free translation systems can also be employed using RNA derived from the DNA of interest. Methods for cell free translation are known to those skilled in the art. (Davis et al., 1986; Ausubel et al., 1992).

Host cells are grown under appropriate conditions to a suitable cell density. If the sequence of interest is operably linked to an inducible promoter, the appropriate environmental alteration is made to induce expression. If the protein accumulates in the host cell, the cells are harvested by, for example, centrifugation or filtration. The cells are then disrupted by physical or chemical means to release the protein into the cell extract from which the protein can be purified. If the host cells secrete the protein into the medium, the cells and medium are separated and the medium retained for purification of the protein.

Larger quantities of protein can be obtained from cells carrying amplified copies of the sequence of interest. In this method, the sequence is contained in a vector that carries a selectable marker and transfected into the host cell or the selectable marker is co-transfected into the host cell along with the sequence of interest. Lines of host cells are then selected in which the number of copies of the sequence have been amplified. A number of suitable selectable markers will be readily apparent to those skilled in the art. For example, the dihydrofolate reductase (DHFR) marker is widely used for co-amplification. Exerting selection pressure on host cells by increasing concentrations of methotrexate can result in cells that carry up to 1000 copies of the DHFR gene.

Proteins recovered can be purified by a variety of commonly used methods, including, but not limited to, ammonium sulfate precipitation, immuno precipitation, ethanol or acetone precipitation, acid extraction, ion exchange chromatography, size exclusion chromatography, affinity chromatography, high performance liquid chromatography, electrophoresis, thin layer chromatography, and ultra filtration. If required, protein refolding systems can be used to complete the configuration of the protein.

The wax ester synthase/DGAT protein encoded by the polynucleotide of SEQ ID NO:1 has been shown to catalyze the transfer of an acyl chain from fatty acyl-CoA to fatty alcohol via condensation of the fatty alcohol with the acyl-CoA and gave DGAT activity, thereby resulting in the formation of a wax ester (see Examples below) in several bacterial strains into which this sequence has been recombinantly introduced. Therefore, in one embodiment, a polynucleotide provided by the invention is employed to produce wax ester in bacteria at a relatively affordable cost. This wax ester, in turn, may be utilized in connection with a number of products including cosmetics, industrial lubricants, coatings, food products, livestock feed, and fermentation media. Livestock includes, but is not limited to, for example, sheep, mules, hogs, cattle, horses, and other grazing animals, or animals commonly raised for agricultural or food production purposes. Fermentation media includes, but is not limited to, for example, a growth media containing, a saccharide, such as glucose, a nitrogen source, a phosphorous source, and agar or other non-digestible polysaccharide, in which the processes of fermentation can occur.

As used in reference to a wax ester synthase protein of the present invention, the term “biological function” or “biological activity” refers to the ability of a wax ester synthase protein to catalyze the transfer of an acyl chain from fatty acyl-CoA to fatty alcohol via condensation of the fatty alcohol with the acyl-CoA, as well as the bifunctional DGAT activity, thereby resulting in the formation of a wax ester.

As used herein, the terms “complementary” or “complementarity” refer to the pairing of bases, purines and pyrimidines, that associate through hydrogen bonding in double stranded nucleic acid. The following base pairs are complementary: guanine and cytosine; adenine and thymine; and adenine and uracil. As used herein the terms include complete and partial complementarity.

As used herein, the term “hybridization” refers to a process in which a strand of nucleic acid joins with a complementary strand through base pairing. The conditions employed in the hybridization of two non-identical, but very similar, complementary nucleic acids vary with the degree of complementarity of the two strands and the length of the strands. Thus the term contemplates partial as well as complete hybridization. Such techniques and conditions are well known to practitioners in this field and further described herein.

As used herein, the term “amino acid” is used in its broadest sense, and includes naturally occurring amino acids as well as non-naturally occurring amino acids, including amino acid analogs and derivatives. The latter includes molecules containing an amino acid moiety. One skilled in the art will recognize, in view of this broad definition, that reference herein to an amino acid includes, for example, naturally occurring proteogenic L-amino acids; D-amino acids; chemically modified amino acids such as amino acid analogs and derivatives; naturally occurring non-proteogenic amino acids such as norleucine, -alanine, ornithine, etc.; and chemically synthesized compounds having properties known in the art to be characteristic of amino acids.

As used herein, the term “proteogenic” indicates that the amino acid can be incorporated into a peptide, polypeptide, or protein in a cell through a metabolic pathway.

As used herein, “expression cassette” means a genetic module comprising a gene and the regulatory regions necessary for its expression, which may be incorporated into a vector.

As used herein, “secretion sequence” or “signal peptide” or “signal sequence” means a sequence that directs newly synthesized secretory or membrane proteins to and through membranes of the endoplasmic reticulum, or from the cytoplasm to the periplasm across the inner membrane of bacteria, or from the matrix of mitochondria into the inner space, or from the stroma of chloroplasts into the thylakoid. Fusion of such a sequence to a gene that is to be expressed in a heterologous host ensures secretion of the recombinant protein from the host cell.

As used herein, a “recombinant nucleic acid” is defined either by its method of production or its structure. In reference to its method of production, e.g., a product made by a process, the process is use of recombinant nucleic acid techniques, e.g., involving human intervention in the nucleotide sequence, typically selection or production. Alternatively, it can be a nucleic acid made by generating a sequence comprising fusion of two fragments which are not naturally contiguous to each other, but is meant to exclude products of nature, e.g., naturally occurring mutants. Thus, for example, products made by transforming cells with any unnaturally occurring vector is encompassed, as are nucleic acids comprising sequences derived using any synthetic oligonucleotide process. Such is often done to replace a codon with a redundant codon encoding the same or a conservative amino acid, while typically introducing or removing a sequence recognition site. Alternatively, it is performed to join together nucleic acid segments of desired functions to generate a single genetic entity comprising a desired combination of functions not found in the commonly available natural forms. Restriction enzyme recognition sites are often the target of such artificial manipulations, but other site specific targets, e.g., promoters, DNA replication sites, regulation sequences, control sequences, or other useful features may be incorporated by design.

As used herein, “polynucleotide” and “oligonucleotide” are used interchangeably and mean a polymer of at least 2 nucleotides joined together by phosphodiester bonds and may consist of either ribonucleotides or deoxyribonucleotides.

As used herein, “sequence” means the linear order in which monomers occur in a polymer, for example, the order of amino acids in a polypeptide or the order of nucleotides in a polynucleotide.

As used herein, “peptide” and “protein” are used interchangeably and mean a compound that consists of two or more amino acids that are linked by means of peptide bonds.

As used herein “recombinant protein” means that the protein, whether comprising a native or mutant primary amino acid sequence, is obtained by expression of a gene carried by a recombinant DNA molecule in a cell other than the cell in which that gene and/or protein is naturally found. In other words, the gene is heterologous to the host in which it is expressed. It should be noted that any alteration of a gene, including the addition of a polynucleotide encoding an affinity purification moiety to the gene, makes that gene unnatural for the purposes of this definition, and thus that gene cannot be “naturally” found in any cell.

As used herein, “targeting sequence” means in the context of gene or polynucleotide insertion, a sequence which results in the gene or polynucleotide being inserted at a particular location by homologous recombination. In the context of proteins or peptides, “targeting sequence” refers to a nucleotide sequence encoding an amino acid sequence the presence of which results in a protein being directed to a particular destination within a cell.

As used herein, “upstream region” means a segment of a polynucleotide that is 5′ to a point of reference on the same polynucleotide.

As used herein, “downstream region” means a segment of a polynucleotide that is 3′ to a point of reference on the same polynucleotide.

As used herein, the terms “construct” and “vector” are used interchangeably.

The detailed description set-forth above is provided to aid those skilled in the art in practicing the present invention. Even so, this detailed description should not be construed to-unduly limit the present invention as modifications and variation in the embodiments discussed herein-can be made by those of ordinary skill in the art without departing from the spirit or scope of the present inventive discovery.

All publications, patents, patent applications and other references cited in this application are herein incorporated by reference in their entirety as if each individual publication, patent, patent application or other reference were specifically and individually indicated to be incorporated by reference.

Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

EXAMPLES Example 1 Materials and Methods

A. Bacterial Strains, Plasmids, Media and Growth Conditions

The bacterial strains and plasmids used in this study are listed in Table 1. Cells of A. calcoaceticus were cultivated aerobically in Luria-Bertani (LB) medium. (Sambrook et al., 2001) in Erlenmeyer flasks without baffles at 30° C. For the induction of wax ester formation cells were cultivated in mineral salts medium (MSM) (Schlegel et al., 1961) with 0.1 g 1⁻¹ NH₄Cl and 1% (w/v) sodium gluconate as carbon source. These culture conditions are referred to as “storage conditions”. Cells of E. coli were grown at 37° C. in LB medium. P. citronellolis was cultivated in LB medium at 30° C. Solidified media contained 1.8% (w/v) agar. Antibiotics were added at the following concentrations if appropriate: ampicillin (Ap) 75 μg ml⁻¹, nalidixic acid (Ndx) 10 μg ml⁻¹, kanamycin (Km) 50 μg ml⁻¹, tetracycline (Tc) 12.5 μg ml⁻¹.

TABLE 1
Bacterial strains and plasmids used in this study

Bacterial strain
or plasmid	Relevant characteristics	Source or reference
Bacteria:
Acinetobacter
calcoaceticus
BD413	unencapsulated mutant of BD4;	ATCC 33305; Juni and
	wax+	Janik, 1969
BD413 (Ndxr)	spontaneous Ndxr mutant of BD413	This study
ACM7	miniTn10Km-induced wax−	This study
	mutant of BD413 (Ndxr)
BD413waxΩKm	wax knock-out mutant of BD413	This study
Escherichia coli
XL1-Blue	recA1, endA1, gyrA96, thi-1,	Bullock, 1987
	hsdR17, (r−k
	m− 7k), supE44, relA1, λ−, lac−
	[F′proABlaclqZΔMS, Tn10(tet)]
S17-1	recA; harbours the tra genes of	Simon et al., 1983,
	plasmid
	RP4 in the chromosome; proA, thi-1
SM10(Apir)	thi-1, thr, leu, tonA, lacY, supE,	Miller and Mekalanos,
	recA::RP4-2-Tc::Mu, Kmr, λpir	1988,
Pseudomona	Wild-type, wax−	DSM 50332
citronellolis
Plasmids:
PLOFKm	MiniTn10Km delivery plasmid, Apr,	Herrero et al., 1990,
	Kmr oriR6K, mobRP4
PHC79	Cosmid, Apr, Tcr	Hohn and Collins,
		1980
PHC79: E8	MiniTn10Km-harbouring EcoR1	This study
	fragment
	24
	from ACM7
Pbluescript SK′	Apr, lacPOZ; T7 and T3 promoter	Stratagene
Pbluescript KS−	Apr, lacPOZ; T7 and T3 promoter	Stratagene
PSK: waxEB19	PCR ™ -amplified 1.9-kbp BamHI-	This study
	EcoRI
	fragment comprising wax in
	pBluescript
	SK−
PKS: waxEB19	PCR ™ -amplified 1.9-kbp BamHI-	This study
	EcoRI
	fragment comprising wax in
	pBluescript
	KS−
PSKsymΩKm	ΩKm in pSKsym	Overhage et al., 1999
	wax disrupted by insertion of ΩKm	This study
	in
	pBluescript SK−
PKS: wax	PCR ™ -amplified 1.5-kbp wax with	This study
	sequence in pBluescript KS−
PKS: wax-His6C	PCR ™ -amplified 1.5-kbp wax with	This study
	S/D
	sequence and C-terminal His6-tag in
	pBluescript KS−
PSER200-4	A. calcoacefcus expression vector;	Reiser and Somerville, 1997
	Kmr
PSER200-4; wax	PCR ™ -amplified 1.9-kbp BamHI-	This study
	EcoRI
	fragment comprising wax in
	pSER200-4
PBBRIMCS-2	broad host range, Kmr, IacPOZ	Kovach et al., 1995
PBBRIMCS-2: wax	PCR ™-amplified 1.5-kbp wax with	This study
	S/D
	Sequence in pBBRIMCS-2

B. miniTn10Km Mutagenesis

miniTn10Km-induced mutants of A. calcoaceticus BD413 (Ndxr) were created according to Herrero et al. (1990) employing the transposon delivery suicide plasmid pLOFKm which was transferred from E. coli SM10 (λpir) to A. calcoaceticus BD413 (Ndx^r) by conjugation by the spot mating technique. Mixtures of donor and recipient were spotted at a 1:1 ratio on LB agar plates containing 50 μM isopopyl-β-D-thiogalactopyranoside (IPTG) for induction of the ISIO_R transposase, which is controlled by the ptac promoter, and were incubated for 16 h at 30° C. Cells were suspended in 10 mM MgSO₄, and appropriate dilutions were plated on MSM plates containing 1% (w/v) sodium gluconate, 50 μg Km ml⁻¹ and 10 μg Ndx ml⁻¹ for selection of transposon-insertion mutants of A. calcoaceticus BD413 (Ndx^r) excluding the presence of auxotrophic mutants.

C. Mutant Screening with Sudan Black B Staining

Mutants of A. calcoaceticus BD413 (Ndx^r) defective in the accumulation of wax esters were identified in accordance to Reiser and Somerville (1997). Mutagenized cells were replica plated onto MSM plates containing 1% (w/v) sodium-gluconate and on LB master plates and were incubated for 48 h at 30° C. to induce wax ester accumulation. The cells on the MSM agar plates were then stained by irrigating the plates-with a 0.02% (w/v) Sudan Black B solution in 50:4:5 (v/v/v) dimethyl sulfoxide-ethanol-water and gently shaking them for 30 min. After this step, the staining solution was disposed, and the plates were carefully washed by gently shaking them for 5 min with 70% (v/v) ethanol. Lighter-staining colonies were identified on these plates, and the corresponding colonies from the master plates were subsequently analyzed by thin-layer chromatography (TLC).

D. Thin-Layer Chromatography (TLC)

Cultures of 50 ml MSM with 1% (w/v) sodium gluconate were inoculated with 3 ml of an overnight LB preculture of A. calcoaceticus and incubated for 24 h at 30° C. Cells were harvested by centrifugation and lyophilized. Neutral lipids were isolated from the cells by extracting 1.5 mg lyophilized cell material with 100 μl chloroform/methanol (1:1, v/v) followed by centrifugation at 13,000 rpm for 2 min. 50 μl of the organic phase were spotted onto a silica gel 60 TLC plate (Merck, Darmstadt, Germany) and the neutral lipids were separated by developing the plates in hexane:diethylether:acetic acid (90:15:1, v/v/v). Lipid spots were visualized by spraying the plates with 40% (v/v) sulfuric acid and charring over a Bunsen flame or by exposition to iodine vapor.

E. Isolation and Manipulation of DNA

Chromosomal DNA of miniTn10Km-induced mutants of A. calcoaceticus BD413 (Ndx^r) was isolated by the method of Marmur (1961). Plasmid DNA was isolated by the method of Bimboim and Doly (1979). DNA restriction fragments were purified from agarose gels using the Nucleotrap-Kit (Macherey-Nagel,Diiren, Germany) following the instructions provided by the manufacturer. Restriction enzymes, T4-ligase and other DNA-manipulating enzymes were purchased from GibcoBRL (Karlsruhe, Germany) and used according to the manufacturer's instructions.

F. Transfer of DNA

Competent cells of E. coli were prepared and transformed by the CaCl₂ procedure as described by Hanahan (1983). Transduction of genomic DNA of A. calcoaceticus BD413, which was ligated into cosmid pHC79 DNA, to E. coli S 17-1 was done as described by Hohn and Murray (1977) after in vitro packaging into λ phages employing the Gigapack III Gold packaging extract (Stratagene, Heidelberg, Germany). Conjugation of E. coli S 17-1 (donor) harbouring hybrid plasmids and P. citronellolis (recipient) was performed on solidified NB medium as described by Friedrich et al. (1981). A. calcoaceticus was transformed as described by Palmen et al. (1993) utilizing the high natural competence of this strain.

G. Genotypic Characterization of the miniTn10 km-Insertion Mutants of A. calcoaceticus BD413 (Ndx^r)

Genomic DNA of miniTn10Km-insertion mutants was digested with EcoRI, and the genomic EcoRI fragments were ligated to cosmid pHC79 DNA. After in vitro packaging in; λ phages the recombinant cosmids were transduced into E. coli S17-1. Recombinant E. coli clones were selected by their Km resistance conferred by the miniTn10Km insertion. The hybrid cosmids were isolated, digested with EcoRI and ligated into the plasmid pBluescript SK. The recombinant plasmids were transformed into E. coli XL1-Blue, and clones resistant to Km plus Ap were selected. The resulting hybrid plasmids were isolated and digested with EcoRI and NotI, which cuts up- and downstream of the Km resistance gene being part of miniTn10Km (Herrero et al. 1990). The resulting fragments were subcloned into EcoRI and NotI digested pBluescript SK and transformed into E. coli XL1-Blue. The obtained hybrid plasmids contained a EcoRI-NotI fragment which included IS10_R or IS10_L, respectively, plus genomic DNA adjacent to the miniTn10Km insertion. The miniTn10Km insertion locus was determined by DNA sequence analysis of the recombinant plasmids using sequencing primers specific to pBluescript SK.

H. DNA Sequencing and Sequence Data Analysis

The dideoxy chain-termination method (Sanger et al., 1977) was used to determine the DNA sequence employing the Sequi Therm EXCEL TM II long-read cycle sequencing kit (Biozym, Hessisch Oldendorf, Germany) and - - - :IRD800-labelled oligonucleotides (MWG-Biotech, Ebersberg, Germany). The primer hopping strategy (Strauss et al., 1986) was applied. Sequencing was performed with a LI-COR DNA model 4000L automatic sequencer (MWG-Biotech, Ebersberg, Germany). Sequence data were compared with sequences deposited in the GeneBank database (online available at www.ncbi.nlm.nih.gov) using the program BlastSearch 2.0.10. (Altschul et al., 1997). Preliminary sequence data from the A. calcoaceticus BD413 genome project were obtained online from www.genoscope.fr and analyzed with the online program pack Biology WorkBench 3.2 at workbench.sdsc.edu.

I. PCR™ Amplifications

PCR™ amplifications of plasmid or genomic encoded DNA were performed according to Sambrook et al. (2001) in a PCR™ Sprint thermocycler (Hybaid, Teddington, UK) with Platinum Pfx DNA polymerase (GibcoBRL, Karlsruhe, Germany).

J. Cloning of wax/dgat and Functional Heterologous Expression

The coding region of the wax/dgat gene including the up- and downstream regions was amplified by tailored PCR™ from genomic DNA of A. calcoaceticus BD413 applying the following oligonucleotides: 5′-AAAGAATTCTGGCCTACATGCAGGCAACTTAA-3′ (5′ end) (SEQ ID NO:5) and 5′-TTTGGATCCGAATTTTCAATACTAGGTACACA-3′ (3′ end) (SEQ ID NO:6) introducing EcoRI and BamHI restriction sites (underlined), respectively. The obtained 1908-bp PCR™ product, shown in FIG. 5 (SEQ ID NO:3), was cloned into EcoRI and BamHI restricted pBluescript KS collinear to the lacZ promoter, resulting in pKS:waxEB 19. Additionally, the obtained 1908-bp PCR™ product was cloned into EcoRI and BamHI restricted pSER200-4, resulting in pSER200-4:wax.

The coding region of the wax/dgat gene without upstream region was amplified by tailored PCR™ from genomic DNA of A. calcoaceticus BD413 applying the following oligonucleotides: 5′-AAAGAATTCAAGGAGGTATCCACGCTATGCGCCCATTAC-3′ (5′ end) (SEQ ID NO:7) introducing a EcoRI restriction site (underlined) and a ribosome binding site (double underlined) and 5′-TTGGATCCAGGGCTAATTTAGCCCTTTAGTT-3′ (3′ end) (SEQ ID NO:8) introducing a BamHI restriction site (underlined). The obtained 1470-bp PCR™ product, shown in FIG. 6 (SEQ ID NO:2), was cloned into EcoRI and BamHI restricted pBluescript KS collinear to the lacZ promoter, resulting in pKS:wax. Additionally, the 1470-bp PCR™ product was cloned into EcoRI and BamHI restricted pBBRIMCS-2 collinear to the lacZ promoter, resulting in pBBRIMCS-2:wax.

For amplification of a DNA fragment encoding a C-terminal His₆-tagged wax ester-synthase by tailored PCR™ using pKS:wax as template, the following oligonucleotides were applied: 5′-AAAGAATTCAAGGAGGTATCCACGCTATGCGCCCATTAC-3′ (5′ end) (SEQ ID NO:9) introducing a EcoRI restriction site (underlined) and a ribosome binding site (double underlined) and ′-TTTGGATCCTTAGTGGTGGTGGTGGTGGTGATTGGCTGTTTT AATATCTTCCT-3′ (3′ end) (SEQ ID NO:10) introducing a BamHI restriction site (underlined) and the His6-tag. The obtained 1430-bp PCR™, product was cloned into EcoRI and BamHI restricted pBluescript KS″ collinear to the lacZ promoter, resulting in pKS:wax-His₆C.

Cells of E. coli harbouring pBluescript KS, pBBRIMCS-2, pKS:waxEB19, pKS:wax, pBBRIMCS-2:wax and pKS:wax-His₆C were cultivated in 50 ml LB medium inoculated with 1% (v/v) of an overnight LB preculture for 6 h at 37° C. in the presence of 1 mM IPTG and appropriate antibiotics. P. citronellolis harbouring pBBRIMCS-2 and pBBR1MCS-2:wax was cultivated in 50 ml LB medium inoculated with 1% (v/v) of an overnight LB preculture for 6 h at 37° C. in the presence of 50 μg Km ml⁻¹. Cells were harvested by centrifugation (10 min, 4,500 rpm at 4° C.), washed with 125 mM sodium phosphate buffer (pH 7.4) and resuspended in 1 ml of the same buffer. Crude extracts were obtained as described below.

50 ml MSM according to Schlegel et al. (1961) were inoculated with 2 ml of LB precultures of P. citronellolis DSM 50332 harbouring pBBRIMCS-2 and pBBRIMCS-2:wax, respectively, and incubated for 48 h at 30° C. with 0.3% (w/v) hexadecanol and 0.3% (w/v) hexadecanol plus 0.5% (w/v) gluconate as carbon sources. Cells were harvested by centrifugation, lyophilized and analyzed for the production of wax esters by TLC.

K. Inactivation of the Wax/dgat Gene of A. calcoaceticus BD413 by Insertion of ΩKm

For inactivation of the wax/dgat gene by insertion of QKm, the 1908-bp PCR™ product described above was cloned into EcoRI and BamHI restricted pBluescript SIC, resulting in—pSK:waxEB19. This hybrid plasmid was restricted with NruI, which cuts within the wax/dgat gene, and ligated with ΩKm, which was recovered by SmaI digestion of plasmid pSKsymΩKm (Overhage et al., 1999), resulting in the hybrid plasmid pSK:waxΩKm. The disrupted wax/dgat gene was isolated from pSK:waxΩKm by digestion with EcoRI and BaMHI and the linear DNA fragment was transformed to A. calcoaceticus BD413. Transformants were selected on LB plates containing 50 μg Km ml⁻¹. The correct exchange of the wax/dgat gene with the disrupted gene in the obtained knock-out strain A. calcoaceticus. BD413waxΩKm was proven by PCR™ using the oligonucleotide primers 5′-AAAGAATTCAAGGAGGTATCCACGCTATGCGCCCATTAC-3′ (5′ end) (SEQ ID NO:11) and 5′-TTTGGATCCAGGGCTAATTTAGCCCTTTAGTT-3′ (3′ end) (SEQ ID NO:12) resulting in a single PCR™ product with the expected size of 2.5 kbp.

L. Preparation of Crude Extracts and Subcellular Fractions

Cells were disrupted by ultrasonification in a Sonopuls GM 200 (Bandelin, Berlin, Germany) with an amplitude of 16 μm (1 min ml⁻¹). Samples were cooled on ice during ultrasonification. Insoluble and soluble protein fractions were obtained after ultracentrifugation at 35,000 g for 30 min. Protein concentrations were determined by the method of Madford (1976). Bovine serum albumin (BSA) fraction V was used as standard.

M. Termination of Wax Ester Synthase Activity

Cells of E. coli were grown for determination of the wax ester synthase activity in the presence of IPTG or in the case of P. citronellolis without IPTG as described above. Measurement of the wax ester synthase activity in A. calcoaceticus was done with cells grown under storage conditions at 30° C. for 24 h. Crude extracts were obtained as described above. Activity of the wax ester synthase was measured in a total volume of 250 μl containing 3.75 mM 1-hexadecanol, 4.63 mg ml⁻¹ BSA, 10 mM MgCl₂, 4.72 μM 1-¹⁴C-Palmitoyl-CoA (specific activity 1.961 Bq pmol⁻¹) and 125 mM sodium phosphate buffer (pH 7.4). Hexadecanol_and BSA were emulsified by ultrasonification. The assays were incubated at 35° C. for 30 min, and the reactions were stopped by extraction with 500 μl chloroform/methanol (1:1, v/v) for 1 min. After centrifugation the chloroform phase was withdrawn, evaporated to dryness, and 40 μg of chloroform-dissolved unlabeled reference wax ester (cetylpalmitate) were added. The lipids were separated by TLC applying hexane:diethylether:acetic acid (90:15:1, v/v/v) as solvent system. After staining the TLC plate with iodine vapor, the spots corresponding to waxes were scraped from the plates into scintillation vials, mixed with 5 ml of liquid scintillation counting cocktail lipoluma (J. T. Baker, Deventer, Netherlands), and radioactivity was measured using a model LS6500 scintillation counter (Beckmann Instruments, Munich, Germany).

Example 2 Results

A. Isolation of Wax-Negative (Wax) Mutants of A. calcoaceticus BD413 (Ndx^r)

Transposon mutagenesis of an isolated spontaneous Ndx-resistant strain of A. calcoaceticus BD413 was performed to obtain mutants affected in the accumulation of wax esters. A total of 4000 miniTn10Km-induced mutants were obtained and screened by Sudan Black B staining for mutants with a reduced or lacking accumulation of storage lipids. Eight mutants were isolated exhibiting a lighter staining with the lipophilic dye Sudan Black B. TLC analysis of these mutants revealed that all of them were unable to accumulate wax esters under storage conditions but were still able to accumulate triacylglycerols (TAGS) to some extent (FIG. 1). These wax mutants were designated ACM for Acinetobacter calcoaceticus mutants.

B. Molecular Characterization of miniTn10Km-Induced Mutants Defective in Wax Esteraccumulation

To map the insertions of miniTn10Km in these mutants, EcoRI fragments, which conferred resistance to Km, were cloned from genomic niutarit DNA. A 8.4-kbp EcoRI fragment was obtained for all eight mutants. For determination of the insertion sequences the 8.4-kbp EcoRI fragments were NotI digested resulting in a 5.1-kbp and a 1.8-kbp EcoRI-NotI fragment for all mutants (FIG. 2), which were subsequently subcloned into pBluescript SK. DNA sequence analyses of the transposon insertion loci revealed that miniTn10Km had inserted in the identical position in all analyzed mutants which makes it very likely that all mutants constitutes siblings. Therefore, only one mutant (ACM7) was selected for the subsequent detailed analyses. Transposon miniTn10Km had inserted with a 9 by palindromic direct repeat (5′-GCGTATGCG-3′) (SEQ ID NO:13) immediately upstream of an ORF with the start codon ATG being part of the direct repeat. The putative translational product exhibited highest homology (37%) to the hypothetical 48.4-kDa protein Rv3740c from Mycobacterium tuberculosis H37Rv which belongs to a group of conserved hypothetical proteins in this strain (Cole et al. 1998a; Cole et al. 1998b). Further sequence comparison of the translational product to Mycobacterium tuberculosis H37Rv and Arabidopsis thaliana revealed several particularly well conserved stretches of amino acids (amino acids 132–139, 258–263, 267–273, 283–289, 373–379, 380388 in SEQ ID NO:4). These conserved amino acid sequences are HHAXVDGV (SEQ ID NO:16), NDVVLA (SEQ ID NO:17), GALRXYL (SEQ ID NO:18), PLXAMVP (SEQ ID NO:19), ISNVPGP (SEQ ID NO:20), and REPLYXNGA (SEQ ID NO:21), wherein “X” is any amino acid.

By sequence comparison of the miniTn10Km harbouring 8.4-kbp EcoRI fragment with the genome sequence data of A. calcoaceticus BD413, the DNA sequence of the native 6.9-kbp EcoRI fragment was obtained which revealed the molecular organization shown in FIG. 3. The hypothetical ORF was obviously not clustered with any genes whose putative translational products might be involved in the biosynthesis of wax esters (FIG. 3 and Table 2).

TABLE 2
Identified ORFs on the 6.9-kbp genomic EcoRI-fragment from A.
calcoaceticus BD413 harboring the wax gene

		Identical
Gene designation	Highest homology to	amino acids
MreC	Rod-shape determining protein MreC	36%
	From P. fluorescens
Maf	Putative inhibitor of septum	45%
	Formation Maf from Salmonella
	Typhimurium LT2
axial filament	Cytoplasmic axial filament protein	58%
	PA4477 from P. aeruginosa PAO1
Wax	Hypothetical 48.4-kDa protein	37%
	Rv3740c from Mycobacterium
	Tuberculosis H37Rv
CysH
	3′-phosphoadenosine-5′-phosphosul-	64%
	Fate reductase (DAPS reductase)
	CysH from P. aeruginosa
thrH (partial)	Homoserine-Kinase ThrH (partial)	68%
	From P. aeruginosa PAO1

C. Inactivation of the ORF (Wax) from A. calcoaceticus BD413 Adjacent to the miniTn10Km Insertion in Wax Mutants

Since the miniTn10Km insertion mapped 5 by upstream of the ATG start codon of an hypothetical ORF in the wax” mutants but leaving the gene itself intact, the phenotype of a mutant with a defective ORF was unknown. Therefore, ORF was disrupted by insertion of the ΩKm gene and a knock-out strain of A. calcoaceticus BD413 was generated as described in Materials and Methods. The obtained strain A. calcoaceticus BD413waxΩKm was analyzed by TLC for its ability to accumulate wax esters under storage conditions. Inactivation of the hypothetical ORF led to the loss of wax ester accumulation whereas TAGs were still produced to some extent (FIG. 4). Thus, the knock-out strain exhibited the same phenotype than the miniTn10Km-induced mutants. Attempts to complement A. calcoaceticus BD413waxΩKm chemically by feeding with the precursor substrates hexadecanal and hexadecanol failed in reconstituting wax ester biosynthesis indicating that the inactivation of the hypothetical ORF probably did not affect the biosynthesis of precursors. A 1.9-kbp fragment comprising the ORF was PCR™ amplified and cloned as a BamHI-EcoRI fragment into pSER200-4which allows constitutive low-level expression in A. calcoaceticus. (Reiser, 1996) resulting in pSER200-4:wax. A. calcoaceticus BD413 harbouring pSER200-4:wax overexpressing the ORF exhibited a twofold higher wax ester synthase activity than the wild-type (Table 3) but the amount of accumulated wax esters was unaltered as estimated by TLC (FIG. 4). Wax ester synthase activity in the wax mutants A. calcoaceticus ACM7 and A. calcoaceticus BD413waxΩKm dropped to only 1% of the wild-type level (Table 3).

TABLE 3
Wax ester synthase activity in crude extracts of different strains of A.
calcoaceticus, E. coli and P. citronellolis.
Values are mean values of experiments done in triplicate.

		Wax ester synthase activity
	Strain	[pmol (mg protein)−1 min−1 ]

	A. calcoaceticus
	BD413	101.7
	ACM7	1.0
	BD413waxΩKm	1.4
	BD413 (pSER200-4)	82.6
	BD413 (pSER200-4: wax)	199.4
	E. coli
	XL1-Blue (pBluescript KS−)	0.3
	XL1-Blue (pKS: wax)	117.6
	XL1-BIue (pKS: wax-His6C)	20.0
	S17-1 (pBBRIMCS-2)	0.5
	S17-1 (pBBRIMCS-2: wax)	128.8
	P. citronellolis
	pBBRIMCS-2	0.5
	pBBRIMCS-2: wax	149.7

D. Cloning and Heterologous Expression of the Wax/dgat Gene

From the results presented above it was concluded that the hypothetical ORF could possibly code for the wax ester synthase, which was therefore designated as wax. Thus, the coding region of ORF plus its up and downstream regions was amplified by PCR™, and the obtained 1908 bp PCR™ product was cloned into EcoRI and BamHI restricted pBluescript KS collinear to the lacZ promoter, resulting in pKS:waxEB 19. However, no wax ester synthase activity could be detected in crude extract IPTG-induced cells of recombinant E. coli XL1-Blue harboring pKS:waxEB 19. Since this could be due to the fact that the putative ribosome binding site (5′-GAGG-3′) (SEQ ID NO:14) 11 bp upstream of the ATG start codon of the ORF (FIG. 5) was not recognized in E. coli, a truncated fragment was amplified by tailored PCR™ introducing a ribosome binding site for E. coli (5′-AAGGAGGT-3′) (SEQ ID NO:15) 9 bp upstream of the ATG start codon(FIG. 6, SEQ ID NO:2), which was cloned as a BamHI-EcoRI fragment into pBluescript KS and pBBRIMCS-2, resulting in the construction of pKS:wax and pBBRIMCS-2:wax. These hybrid plasmids were transformed into E. coli XL1-Blue and E. coli S17-1, respectively, and wax ester synthase activity was measured in crude extracts of IPTG-induced cultures. The wax gene was functionally heterologously expressed in E. coli XL1-Blue (pKS:wax) as well as in E. coli S17-1 (pBBRIMCS-2:wax) resulting in an active wax ester synthase with activities of 117.6 and 128.8 pmol (mg protein min)⁻¹, respectively, whereas in the control strains harboring only the vectors activities of only 0.3 and 0.5 pmol (mg protein min)⁻¹, respectively, were determined (Table 3). The enzyme activity was almost equally distributed between the insoluble and soluble fraction of the crude extracts (data not shown). These data clearly show that the wax/dgat gene encodes for an active wax ester synthase. The activity in recombinant E. coli strains harboring the wax gene was even higher than in the origin strain A. calcoaceticus BD413 25 (Table 3).

Additionally, pBBRIMCS-2:wax was transferred conjugatively to P. citronellolis, a Gram-negative alkane degrading bacterium unable to accumulate wax esters. Also in this host the wax gene was expressed constitutively resulting in a wax ester synthase activity of 149.7 pmol (mg protein min)−1, whereas the control harboring only the vector exhibited an activity of only 0.5 pmol (mg protein min)−1. (Table 3).

Numerous Pseudomonas strains have been reported to be able to utilize n-alkanes as sole carbon source (Baptist et al., 1963; Macham and Heydeman, 1974; Williams et al., 1981). Alkane degradation route proceeds via successive terminal oxidations leading to the formation of the corresponding fatty alcohols, fatty aldehydes and fatty acids, which are subsequently esterified to the respective fatty acyl-CoA thioesters and channeled into the β-oxidation cycle (Baptist et al., 1963). The inventors tested a strain of P. citronellolis, for which the inventors have demonstrated heterologous expression of the A. calcoaceticus BD413 wax/dgat gene in a functionally active form (see Table 3), for its ability to utilize long-chain n-alkanes. This strain was able to grow on n-alkaes with chain-length from C 10 up to C 16 as sole carbon source (n-alkaes with chain-lengths longer than C 16 were not -tested) (data not shown). In addition, this strain could utilize also hexadecanol as an intermediate of the alkane degradation pathway as sole carbon source (data not shown). In P. citronellolis harbouring pBBRIMCS-2:wax, the cultivation in MSM with 0.3% (w/v) hexadecanol as sole carbon source led to the formation of small but significant amounts of wax esters as revealed by TLC analysis, whereas no wax esters were detectable in the control strain harbouring only the vector (FIG. 7). The uptake of hexadecanol into the cells, its oxidation to fatty acids via the alkane degradation pathway and their subsequent metabolization to acyl-CoA provided obviously sufficiently high intracellular levels of substrates of the wax ester synthase allowing the production of wax esters in the strain expressing the wax ester synthase from A. calcoaceticus BD413. However, no wax esters were formed when the cells were co-cultivated with 0.3% (w/v) hexadecanol plus 0.5% (w/v) gluconate (FIG. 7). Alkane oxidation activity has been reported to be subjected to carbon catabolite repression for various strains of P. aeruginosa (Dalhoff and Rehm, 1976; van der Linden, 1963; van Eyk and Bartels, 1968; P. putida (Fish et al., 1982; Grund et al., 1975; P. oleovorans (Staijen et al., 1999). The presence of gluconate has probably repressed the alkane degradation system in P. citronellolis. Therefore, no acyl-CoA thioesters could be formed from hexadecanol via the alkane degradation pathway during co-cultivation on 0.3% (w/v) hexadecanol plus 0.5% (w/v) gluconate, and thus the intracellular acyl-CoA level was to low to promote significant wax ester production in the strain expressing the wax ester synthase of A. calcoaceticus BD413 under this conditions.

E. Generation of His_b-Tagged Wax Ester Synthase and Purification of the Enzyme

For the purpose of the purification of the wax ester synthase enzyme, pKS:wax-His₆C was constructed as described in Materials and Methods resulting in the expression of a C-terminal His₆-tagged protein. However, recombinant E. coli XL1-Blue (pKS:wax-His₆C) expressing the C-terminal His₆-tagged wax ester synthase exhibited only 17% activity in comparison to E. coli XL1-Blue harbouring the native wax/dgat gene on pKS:wax (Table 3). An attempt to purify the C-terminal His₆ tagged wax ester synthase chromatographafically using the Ni-NTA Spin Kit (Qiagen, Hilden, Germany) revealed only a very weak binding of the native enzyme on-the Ni-NTA column, resulting in an only 4.3-fold enrichment (data not shown). This indicates that the C-terminus of the protein is obviously not localized on the surface of the native enzyme but in the interior, which could explain that the addition of the 6 histidine residues to the C-terninus of the protein has such a strong negative effect on the enzyme activity.

Example 3 The Wax Gene Encodes a Bifunctional WS/DGAT

Wild-type A. calcoaceticus ADP1 exhibited a WS activity of 90.37 pmol (mg min)−1 and a ca. tenfold lower DGAT activity (Table 4), which corresponded approximately with the amounts of wax esters and TAGs accumulated under storage conditions as estimated by TLC. Inactivation of wax/dgat not only caused the loss of the ability for wax ester and TAG biosynthesis; it also abolished WS and DGAT activity in the transposon-induced mutant as well as in the knock-out mutant (Table 4).

TABLE 4
Shows WS and DGAT activities in crude cell extracts of different strains.
Values are averages of at least three independent studies.

			WS activity	DGAT activity
Strain		Plasmid	[pmol (mg min)−1]	[pmol (mg min)−1]

A. calcoaceticus	ADP1		90.37	7.96
	ACM7		0.20	0.11
	ADP1wax/dgat		0.65	0.17
	ΩKm
E. coli	XL1-Blue	pBluescript KS−	0.11	0.11
		pKS: wax/dgat	84.51	9.37
		pBluescript SK−	0.10	0.10
		pSK: wdh3269	0.65	0.31
	S17-1	pBBRIMCS-2	0.11	0.10
		pBBRIMCS-2: wax/dgat	99.20	25.03
P. citronellolis		pBBRIMCS-2	0.10	0.10
		pBBRIMCS-2: wax/dgat	99.20	25.03
P. citronellolis		pBBRIMCS-2	0.10	0.10
M. smegmatis	mc2155		106.54	96.44
R. opacus	PD630	pBBRKmNC903	9.59	6.66
		pBBRLKmNC903-	12.29	10/73
		PSK::wdh3269

Heterologous expression of wax/dgat conferred the capability to recombinant E. coli XL1-Blue harbouring pKS:wax/dgat to catalyze the acyl-CoA-dependent acylation of fatty alcohol as well as of DAG (FIG. 2A) at rates similar to those of A. calcoaceticus ADP1 (Table 4). These results clearly show that both WS and DGAT activity arise from wax/dgat, which therefore codes for a bifunctional WS/DGAT enzyme. Furthermore, the studies with the knock-out mutant indicate that no other protein exhibiting WS or DGAT activity contributes significantly to wax ester or TAG biosynthesis in A. calcoaceticus ADP1. This was supported by the fact that no other wax/dgat homologue could be identified by BLAST (Altschul et al., 1997) search in the preliminary A. calcoaceticus ADP1 genome sequence data accessible online at www.genoscope.fr. However, residual trace amounts of TAGs accumulated in the mutants indicate the presence of a minor alternative pathway which is, however, only active at a very low-rate. Functional heterologous expression of wax/dgat was not only demonstrated in E. coli S17-1 but also in Pseudomonas citronellolis (Table 4).

The bifunctional WS/DGAT comprises 458 amino acids with a theoretical molecular weight of 51.8 kDa and a pI of 9.05. It is a rather amphiphilic protein, and it possesses one putative predicted membrane-spanning region (FIG. 12B).

Heterologous functional expression of the wax/dgat gene in the alkane-degrading bacterium P. citronellolis resulted in an active enzyme which maintained its bifunctionality (Table 4). During cultivation of P. citronellolis (pBBR1MCS-2:wax/dgat) under storage conditions, no accumulation of wax esters could be detected by TLC if 0.5% (w/v) gluconate, 0.3% (w/v) 1-hexadecane or 0.3% (w/v) palmitate were used as carbon sources (data not shown). However, cultivation on 0.3% (w/v) 1-hexadecanol, which can serve as a direct substrate for the WS, resulted in recombinant production of wax esters (FIG. 13B). No TAG accumulation could be observed under either conditions.

Example 5 Wax Ester Biosynthesis in M. smegmatis mc²155.

Eight WS/DGAT homologous genes could be identified in a preliminary genome sequence of the non-pathogenic strain M. smegatis mc²155 (see Table 6), which is publicly accessible online via NCBI. The gene with the highest similarity exhibits 41.0% amino acids identity to WS/DGAT and 67.2% to the hypothetical protein Rv3734c from M. tuberculosis H37Rv (Table 5). Recombinant E. coli expressing this gene (designated as wdh3269) on plasmid pSK:wdh3269 showed a weak WS and DGAT activity which was slightly but reproducible higher than the vector control (Table 1).

M. smegmatis mc²155 cultivated under storage conditions with glucose as sole carbon source exhibited both high WS as well as DGAT activity in vitro (Table 4), but in vivo only TAGs were intracellularly accumulated (FIG. 13A). However, like recombinant P. citronellolis, M. smegatis mc²155 was also capable of substantial wax ester biosynthesis in vivo when 1-hexadecanol was provided as sole carbon source or as a co-substrate (FIG. 13A).

TABLE 5
Wax/DGAT related proteins in M. tuberculosis and A. thaliana. Accession
numbers correspond to NCBI protein data base. Identities to the
A. calcoaceticus ADP1 WS/DGAT were based on BLAST search
results and calculated for full-length sequences.

		Length	Identity
Strain	Protein	(amino acids)	(%)	Accession

M. tuberculosis	Rv3740c		448	39.7	NP_218257
H37Rv	Rv3734c	454	38.6	NP_218251
	Rv1425	459	34.9	NP_215941
	Rv3480c	497	34.8	NP_217997
	Rv2285	445	33.2	NP_216801
	Rv0895
	505	31.7	NP_215410
	Rv3088
	474	28.9	NP_217604
	Rv3130c
	463	27.0	NP_217646
	Rv3087	472	26.7	NP_217603
	Rv1760
	502	25.7	NP_216276
	Rv0221
	469	23.9	NP_214735
	Rv2484c	491	22.6	NP_217000
	Rv3371	446	20.7	NP_217888
A. thaliana	At5g53380	483	21.3	NP_200150
	At5g16350	488	20.5	NP_197139
	At5g12420	480	19.8	NP_568275
	At5g22490
	482	18.9	NP_197641
	At1g72110	479	16.5	NP_177356
	At5g37300	481	16.0	NP_568547
	At3g49210	518	15.6	NP_190490
	At3g49190	522	15.5	NP_190488
	At5g53390
	485	15.1	NP_200151
	At3g49200	507	14.4	NP_190489

Example 6 Identification of a Putative Active Site

The A. calcoaceticus ADP1 WS/DGAT and the related proteins in Mycobacterium and A. thaliana exhibit in their N-terminal region some partial similarity to a conserved condensing domain found in many multi-domain enzymes synthesizing peptide antibiotics (NCBI Conserved Domain Database accession pfam00668). This condensing domain contains an active-site motif (HHXXXDG), whose second histidine residue is strictly conserved and has been demonstrated to be essential for catalytic activity in nonribosomal peptide bond formation (Stachelhaus et al., 1998). The WS/DGAT and related proteins also contain this putative active site with the motif (HXXXDG) being strictly conserved (FIG. 10). Thus, it is very likely that this site is catalytically participating in the acyl-CoA acyltransferase reactions involved in wax ester and TAG formation (FIG. 12A).

The motif HHXXXDG corresponding to amino acids 132–138 of the A. calcoaceticus WS/DGAT is highly conserved, and may be the catalytic site responsible for the ester bond formation (FIG. 10). This motif is the putative active site in the condensing domain found in many multi-domain enzymes synthesising peptide antibiotics.

It was shown that M. smegatis mc²155 possesses both DGAT and WS activity in vitro (Table 4), and that this strain can produce TAGs as well as wax esters in vivo (FIG. 13A). Wax ester synthesis from unrelated carbon sources like glucose, however, was hampered probably only by the lacking capability of fatty alcohol biosynthesis. Heterologous expression of the homologue from M. smegatis mc²155, which exhibits the highest similarity to the A. calcoaceticus ADP1. WS/DGAT (wdh3269), mediated low but significant WS and DGAT activities to recombinant E. coli (Table 4); these low activities could result from low expression of the gene. The activities may arise in M. smegmatis mc²155 also from WS/DGAT homologues or may be the cumulative result of two or more enzymes. Alternatively to storage lipid synthesis, some of the WS/DGAT homologues could also participate in biosynthesis of cell wall lipids like mycolic acids, which are responsible for the unique properties of the mycobacterial cell wall and can play crucial roles in pathogenesis.

TABLE 6
Distribution of WS/DGAT related proteins in bacteria.
Data were obtained using BLAST search with microbial genomes at NCBI
comprising 158 eubacterial + 18 archaeal finished and unfinished
genome sequences (as of August 2002). Identities were calculated
for full-length sequences.

	Number of
Strain	related proteins	Highest identity (%)

M. tuberculosis CDC1551	14	39.7
M. tuberculosis H37Rv	13	39.7
M. bovis	13	39.7
M. avium	10	40.4
M. smegmatis mc2155	8	41.0
M. avium subsp. paratuberculosis	8	40.4
S. coelicolor A3(2)	2	26.6
M. leprae	1	18.9

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Claims (21)

1. An isolated polynucleotide encoding the polypeptide of SEQ ID NO:4.

2. An isolated polynucleotide encoding a polypeptide having wax ester synthase/acyl-CoA:diacylglycerol acyltransferase activity, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence that hybridizes to the complement thereof under conditions of 5×SSC, 50% formamide and 42° C.

3. The isolated polynucleotide of claim 2, wherein the polynucleotide encodes the polypeptide encoded by SEQ ID NO:1.

4. The isolated polynucleotide of claim 2, wherein the polynucleotide is isolated from a bacterium.

5. The isolated polynucleotide of claim 4, wherein the bacterium is a gram negative bacterium.

6. The isolated polynucleotide of claim 5, wherein the bacterium is Acinetobacter calcoaceticus.

7. The isolated polynucleotide of claim 2, comprising the nucleic acid sequence of SEQ ID NO:1 or the complement thereof.

8. A recombinant vector comprising an isolated polynucleotide encoding a polypeptide having wax ester synthase activity and having the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence that hybridizes thereto under conditions of 5× SSC, 50% formamide and 42° C.

9. The recombinant vector of claim 8 further comprising at least one additional sequence selected from the group consisting of

(a) a regulatory sequence operatively coupled to the polynucleotide;

(b) a selection marker operatively coupled to the polynucleotide;

(c) a marker sequence operatively coupled to the polynucleotide;

(d) a purification moiety operatively coupled to the polynucleotide;

(e) a secretion sequence operatively coupled to the polynucleotide; and

(f) a targeting sequence operatively coupled to the polynucleotide.

10. The recombinant vector of claim 9, wherein the vector comprises a developmentally-regulated, an organelle-specific, a tissue-specific, an inducible, a constitutive or a cell-specific promoter.

11. The recombinant vector of claim 8, wherein the recombinant vector is selected from the group consisting of pKS:waxEB19, pKS:wax, and pKS:wax-His6C.

12. The recombinant vector of claim 8, wherein the polypeptide has wax ester synthase/acyl-CoA:diacylglycerol acyltransferase (WS/DGAT) activity.

13. The recombinant vector of claim 8, wherein the polynucleotide encodes at least one conserved amino acid sequence selected from the group consisting of HHAXVDGV (SEQ ID NO:16), NDVVLA (SEQ ID NO:17), GALRXYL (SEQ ID NO:18), PLXAMVP (SEQ ID NO:19), ISNVPGP (SEQ ID NO:20), and REPLYXNGA (SEQ ID NO:21).

14. A host cell transformed with the recombinant vector of claim 8.

15. The host cell of claim 14, wherein the host cell is selected from the group consisting of a mammalian cell, plant cell, insect cell, yeast cell, fungus cell, and bacterial cell.

16. The host cell of claim 15, wherein the host cell is selected from the group consisting of E. Coli and Pseudomonas citronellolis.

17. The host cell of claim 14, wherein the host cell expresses a protein encoded by the recombinant vector.

18. The host cell of claim 17, wherein the expressed protein is secreted by the host cell.

19. A method for producing a wax ester comprising culturing a host cell of claim 14 in the presence of at least one substrate of said polypeptide having wax ester synthase activity under conditions permitting expression of the polypeptide having wax ester synthase activity.

20. The method of claim 19, further comprising isolating the wax ester from the host cell or from the medium in which the host cell is cultured.

21. The method of claim 19, wherein the host cell is a bacterial cell.

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